Lipoprotein lipase (LPL), which is essential for utilization of plasma triacylglycerol (TG), is synthesized by parenchymal cells and acts in capillaries where it is anchored by heparan sulfate proteoglycan. Brown adipocytes cultured from newborn mice synthesize LPL which is active, dimerized (M(r) = 110,000) and secreted, and has endo H-resistant, - partially resistant and -totally sensitive oligosaccharide chains, and high affinity for heparin-Sepharose (eluted with 0.9 M NaCl). Combined lipase deficiency (cld) is a recessive mutation in mice which affects post-translational processing, not the structural gene, of LPL. Cultured cld/cld brown adipocytes synthesize LPL which is inactive, not dimerized, and retained in endoplasmic reticulum, and has totally endo H-sensitive chains and low affinity for heparin-Sepharose (eluted with 0.45 M NaCl). It is not known whether the primary defect in cld/cld cells is faulty processing in ER or lack of transport of LPL from ER. Brefeldin A (BFA), which causes relocation of Golgi enzymes into ER, was used to study the effect of Golgi enzymes on LPL retained in ER. LPL activity was increased from 0% to 50% of normal in cld/cld cells treated 4 h with BFA. About one-third of the LPL was dimerized, and one-tenth acquired high heparin-affinity. Most of the LPL subunits in BFA-treated cld/cld cells were processed to partial endo H-resistance. The active LPL in BFA-treated cld/cld cells was dimeric and had high affinity for heparin. The findings indicate that synthesis of inactive LPL in cld/cld cells probably results from impaired transport of LPL from ER. The transport defect may involve a defective LPL-anchoring system, involving heparan sulfate proteoglycan or glycosyl phosphatidylinositol, or a structural change in the LPL molecule preventing interaction with the anchor. Plasma of cld/cld mice has 250 times normal triacylglycerol concentra- tion and one-tenth normal ratio of cholesteryl ester to cholesterol. Most apo E and apo A-IV in plasma of cld/cld mice were present in chylomicrons, whereas apo E in newborn normals was mostly in HDL, and apo A-IV was in VLDL and HDL. Apo A-I in cld/cld mice was equally distributed between chylomicrons and HDL, whereas all apo A-I in normal mice was in HDL. Normal cholesteryl esterification may require release of one or more of the apoproteins retained in cld/cld chylomicrons. Preliminary findings suggest synthesis of apo B-100, a glycoprotein with N-linked oligosaccharides, is impaired in cld/cld mice.